The long-term objectives of this application are to elucidate the mechanisms and regulation of renal tubular calcium transport. The specific aim is to investigate the role of cytosolic free calcium in parathyroid hormone-mediated and -independent calcium reabsorption in the cortical thick ascending limb of Henle's loop and in the connecting tubule, which are two important calcium-reabsorbing segments of the nephron. Young growing rabbits will be fed a calcium-replete or -deficient diet. Individual cortical thick ascending limbs and connecting tubules will be dissected from their kidneys and perfused in vitro. Epithelial calcium transport will be measured with calcium-45 or by continuous-flow microcolorimetry. Cytosolic free calcium concentration will be measured simultaneously in the same perfused tubule segment by quantitative fluorescence microscopy using fura-2 as an intracellular calcium indicator. Perfusion and bathing solution compositions will be manipulated independently and in concert to identify potential plasma membrane calcium transport processes at both the luminal and anti-luminal sides of the cell. The individual effects of luminal and peritubular extracellular fluid calcium concentration on cytosolic free calcium concentration will be assessed, and the role of cytosolic free Ca, whether as a second messenger in response to parathyroid hormone or as part of a transcellular Ca flux, will be determined. The regulation of urinary calcium excretion is important to calcium homeostasis. For example, all mammalian species that have been studied, including the rabbit, markedly reduce calcium excretion when fed a calcium-deficient diet. Although parathyroid homone probably is an important factor in augmenting calcium reasbsorption under these conditions, the mechanisms by which it acts and the nature of parathyroid hormone-independent mechanisms underlying renal tubular conservation of calcium are unknown. The propose studies will provide new information that will advance our knowledge in this area from the epithelial to the cellular level.